Cocaine-induced changes in D(2) receptors
have been implicated in the expression of
sensitized behavioral responses and
addiction-like behaviors; however, the influence
of D(3) receptors is less clear.

OBJECTIVES: To characterize the effects of
repeated cocaine administration on the
sensitivity of rats to D(2)- and D(3)-mediated
behaviors, as well as the binding properties of
ventral striatal D(2)-like and D(3)
receptors.

METHODS: Pramipexole was used to assess the
sensitivity of rats to D(3)/D(2) agonist-induced
yawning, hypothermia, and locomotor activity, 24
h, 72 h, 10, 21, and 42 days after repeated
cocaine or saline administration. The locomotor
effects of cocaine (42 day) and the binding
properties of ventral striatal D(2)-like and
D(3) receptors (24 h and 42 days) were also
evaluated.

RESULTS: Cocaine-treated rats displayed an
enhanced locomotor response to cocaine, as well
as a progressive and persistent leftward/upward
shift of the ascending limb (72 h-42 day) and
leftward shift of the descending limb (42 days)
of the pramipexole-induced yawning
dose-response curve. Cocaine treatment also
decreased B (max) and K (d) for D(2)-like
receptors and increased D(3) receptor binding at
42 days. Cocaine treatment did not change
pramipexole-induced hypothermia or locomotor
activity or yawning induced by cholinergic or
serotonergic agonists.

CONCLUSIONS: These studies suggest that
temporal differences exist in the development of
cocaine-induced sensitization of D(3) and D(2)
receptors, with enhancements of D(3)-mediated
behavioral effects observed within 72 h and
enhancements of D(2)-mediated behavioral effects
apparent 42 days after cocaine. These findings
highlight the need to consider changes in D(3)
receptor function when thinking about the
behavioral plasticity that occurs during
abstinence from cocaine use.

Introduction

Early studies on the effects of repeated
cocaine administration described a common
behavioral sequelae in monkeys, dogs, and rats
in which the behavioral and physiologic effects
of cocaine were observed sooner, and with
greater intensity following repeated,
intermittent cocaine administration (Downs and
Eddy 1932a, b; Post and Rose 1976; Tatum and
Seevers 1929), and the progressive and
persistent nature of these effects, frequently
referred to as behavioral sensitization, are now
well established in rodents. Although some
important differences exist between effects of
experimenter- and self-administered cocaine
(Jacobs et al. 2003), a variety of progressive
and time-dependent neuropharmacologic and
behavioral changes have been shown to correspond
to the expression of the sensitized locomotor
response to cocaine, including decreases in
dopamine transporter (DAT) density and
enhancements of cocaine-induced increases in
extracellular dopamine, regardless of whether
cocaine is experimenter(Heidbreder et al. 1996;
Henry and White 1995; Kalivas and Duffi 1993;
Pilotte et al. 1994; Wilson et al. 1994), or
self-administered (Ahmed and Cador 2006; Ferrano
et al. 2005; Hooks et al. 1994; Phillips and Di
Ciano 1996; Zapata et al. 2003). The repeated
administration of psychostimulants is also known
to affect a variety of neurotransmitter systems
(e.g., cholinergic, dopaminergic, GABAergic,
glutamatergic, opioidergic, and serotonergic),
and it is thought that the neuroadaptations that
underlie the development and expression of the
sensitized behavioral responses are also
important for the development of addiction-like
behaviors in animals (for reviews, see Kalivas
and Stewart 1991; Pierce and Kalivas 1997;
Robinson and Berridge 1993; Vanderschuren and
Kalivas 2000; Vezina 2004). An important theory
of the basis of drug addiction posits that
drug-induced behavioral sensitization reflects
sensitized brain circuits that, in turn, enhance
the incentive value of the drugs and the cues
associated with them (Robinson and Berridge
1993). Among the data that support this theory
are findings that cocaine-induced sensitization
of D2-like (D2, D3, and D4) receptors within the
nucleus accumbens plays a role in the increased
effectiveness of D2-like agonists in inducing
cocaine-directed responding (i.e., drug-seeking)
following the cessation of cocaine
selfadministration (De Vries et al. 2002; Dias
et al. 2004; Edwards et al. 2007). What is not
specified in the theory or demonstrated in these
data are the respective roles of the D2, D3, and
D4 receptors in mediating either the behavioral
sensitization or the corresponding neurological
changes.

Despite these apparent differences in the
effects of cocaine on D2 and D3 receptor levels,
the lack of agonists or antagonists with high
degrees of selectivity for the D2 or D3 receptor
has made it difficult to parse the relative
contributions of these receptor subtypes to the
expression of sensitized behavioral responses or
addiction-like behaviors. One approach that has
been useful in determining the involvement of
different dopamine receptor subtypes has been to
evaluate the capacity of cocaine to sensitize
animals to the selective behavioral effects of
DI-like (D1 and D5) or D2-like (D2, D3, and D4)
receptor agonists. Accordingly, increases in
SKF-3 8393-induced tongue protrusions
(Neisewander et al. 1996) have been interpreted
as a cocaine-induced sensitization of Dl -like
receptors, whereas enhancements of quinpirole-,
or quinelorane-induced sniffing, head movements,
or locomotor stimulation (De Vries et al. 2002;
Dias et al. 2004; Edwards et al. 2007; Ujike et
al. 1990) have been attributed to a
sensitization of D2 receptors. It should be
noted, however, that these latter studies do not
permit a discrimination between effects produced
through the D2 as opposed to the D3 receptors as
the majority of these effects are observed at
doses of quinpirole or quinelorane that are 50-
to 250-fold higher than those required to induce
behaviors that have been attributed to the
selective activation of D3 receptors, such as
yawning (Collins et al. 2007; Collins et
al. 2005), and 5- to 25-fold higher than those
required to produce D2-mediated effects, such as
hypothermia (Chaperon et al. 2003; Collins et
al. 2007).

Support for these sub-type-specific roles of
the D3 and D2 receptors in the regulation of
yawning and hypothermia has been provided
by antagonist interaction studies in which
D3-selective antagonists have been shown to
produce dose-dependent and selective rightward
shifts of the ascending limb of the
dose-response curve for D2-like agonist-induced
yawning, whereas D2-preferring
antagonists have been shown to produce selective
rightward shifts of the descending limb of the
yawning doseresponse curve for D2-like
agonist-induced yawning at doses that
also inhibit the induction of hypothermia
(Baladi et al. 2009; Collins et al. 2008;
Collins et al. 2007; Collins et al. 2009;
Collins et al. 2005). Based on these
differential roles of the D3 (induction) and D2
(inhibition) receptors in the mediation of
yawning, we have hypothesized that
cocaine-induced increases in D3 receptor density
and/or sensitivity would result in leftward
shifts of the ascending limb of the
yawning dose-response curve, whereas
decreases in D2 receptor density would result in
rightward shifts of the descending limb of the
yawning dose-response curve. Conversely,
an increase in the sensitivity of D2 receptors
should result in a leftward shift of the
descending limb of the yawning
doseresponse curve.

In the current experiments, rats were first
given a regimen of once daily cocaine or saline
injections prior to assessing the sensitivity of
rats to the D3- and D2-mediated behavioral
effects of the D3-preferring agonist,
pramipexole (and '-30-fold selective for D3 over
D2 in vivo; Collins et al. 2007; '-90-fold
selective for D3 over D2 in vitro; Millan et al.
2002). Dose-response curves for
pramipexoleinduced yawning, locomotor
activity, and hypothermia were obtained 24 h, 72
h, 10, 21, and 42 days after the cessation of
cocaine or saline administration to allow for
evaluation of the development of cocaine-induced
changes in D3- and/or D2-mediated behavioral
effects. To assess the selectivity of cocaine's
effects for dopaminergic systems, dose-response
curves for yawning induced by the
indirect cholinergic agonist, physostigmine, and
the 5-HT2 receptor agonist,
3-trifluoromethylphenylpiperazin (TFMPP), were
also obtained after 24-72 h and 41-43 days of
cessation from cocaine or saline administration.
In parallel, radioligand binding studies were
performed to assess the effects of repeated
cocaine administration on the binding properties
of ventral striatal D2 and D3 receptors 24 h and
42 days after the cessation of cocaine or saline
administration.

Discussion

In agreement with the majority of previous
studies, cocainetreated rats displayed an
enhanced locomotor response to cocaine 42 days
after the cessation of cocaine, suggesting that
the current regimen of cocaine administration
was sufficient to produce a long-lasting
behavioral sensitization to cocaine. In
addition, although cocaine treatment failed to
alter pramipexole-induced locomotor activity or
hypothermia, cocaine-treated rats displayed a
progressive and persistent leftward and/or
upward shift in the ascending limb of the
dose-response curve for pramipexole-induced
yawning, as well as a leftward shift in
the inhibition of yawning that comprises
the descending limb of the doseresponse curve
for yawning. The temporal differences in
the development of these two effects suggest
that increases in the sensitivity of rats to
D3-mediated behavioral effects become apparent
shortly after the cessation of cocaine with
progressive and persistent enhancements observed
for up to 42 days, whereas increases in the
sensitivity of rats to D2mediated behavioral
effects do not emerge until 42 days after the
cessation of cocaine.

Although it has been well established that
repeated, experimenter- or self-administered
cocaine results in a progressive and persistent
enhancement of the locomotor stimulatory effects
of subsequent cocaine challenges (Heidbreder et
al. 1996; Henry and White 1995; Kalivas and
Duffy 1993), it is important to note that
crosssensitization between the locomotor
stimulatory effects of cocaine and direct
D2-like agonists has been reported with
self-administered cocaine (De Vries et al. 2002;
Dias et al. 2004; Edwards et al. 2007), but not
experimenteradministered cocaine (e.g., Djano
and Martin-Iverson 2000). The inability of
cocaine to cross-sensitize rats to the locomotor
stimulatory effects of pramipexole in the
present studies provides further support for
this dichotomy between experimenter- and
self-administered cocaine. However, it is
possible that cross-sensitization was not
observed due to the relatively low doses of
pramipexole that were used, the relatively short
period of time that pramipexole-induced
locomotor activity was measured, or some
combination thereof (De Vries et al. 2002; Dias
et al. 2004; Edwards et al. 2007).
Alternatively, it has also been suggested that
the development (Henry et al. 1998; Hoflthan and
Wise 1993) and expression (Djano and
Martin-Iverson 2000; Kiyatkin 1994) of
cross-sensitization between the locomotor
stimulatory effects of experimenter-administered
cocaine and D2like agonists depends upon an
activation of both Dr and Dr like receptors,
raising the possibility that basal extracellular
dopamine levels were insufficient to activate
D1-like receptors during the locomotor tests.
Nevertheless, when taken together with previous
reports of food restrictioninduced enhancements
of the locomotor stimulatory effects of
pramipexole under identical experimental
conditions (Collins et al. 2008), the current
fmdings suggest that locomotor assays are
insufficient to detect changes in D2 and/or D3
receptor sensitivity following
experimenteradministered cocaine in rats.

Despite this inability of prior cocaine
administrations to alter the locomotor effects
of pramipexole, cocaine-treated rats displayed a
progressive and persistent leftward and upward
shift of the D3-mediated (Collins et al. 2007;
Collins et al. 2005), ascending limb of the
dose-response curve for pramipexole-induced
yawning, as well as an increased
sensitivity to the D2-mediated (Collins et al.
2007; Collins et al. 2005) inhibitory effects of
pramipexole on yawning when tested after
the cessation of cocaine administration. These
findings not only suggest that yawning
assays are sensitive enough to differentiate
between cocaine-induced changes in the D3- and
Dr mediated behavioral effects of pramipexole,
but also that there are temporal differences in
these effects. Progressive enhancements of
D3-mediated behaviors emerged shortly after the
cessation of cocaine administration ('-72 h) and
persisted for at least 42 days, whereas
increases in the sensitivity of rats to some
D2-mediated behaviors do not become apparent
until after a prolonged period of time since the
cessation of cocaine administration ('-42 days).
Importantly, cocaine treatment failed to alter
the induction of yawning by the indirect
cholinergic agonist, physostigmine, or the 5-HT2
receptor agonist, TFMPP, suggesting that the
cocaine-induced changes in pramipexole-induced
yawning represent changes in the
sensitivity and/or expression of the D3 and D2
receptors that mediate yawning and not a
more general enhancement of the signaling
pathways within the paraventricular nucleus that
regulate yawning.

Although the relatively low levels of D2 and
D3 receptor expression and small volume of the
paraventricular nucleus precluded the ability to
accurately assess changes in the binding
properties of D2-like receptors within this
brain region, the results of radioligand binding
studies performed on ventral striatal tissue
provide molecular evidence to support these
behavioral changes. Importantly, although
3H-spiperone binds to both the D2 and D3
receptor subtypes, its preferential affinity for
D2 compared to D3 receptor ('-80-fold selective
for D2 over D3 in rat brain using the current in
vitro assay conditions; Levant and DeSouza
1993), combined with the differential levels of
expression for the D2 and D3 receptors (D2
expressed at -100-fold higher levels than D3;
Levesque et al. 1992), means that the
cocaine-induced changes in the Bmw. and Kd
obtained with 3H-spiperone in the current
studies can be primarily attributed to changes
in the binding properties of D2 receptors.
Similar to what was observed with
pramipexole-induced yawning at the 24 h
time-point, cocaine treatment failed to alter
the binding properties of D3 or D2-like
receptors at this early time-point. Although
previous studies in mice, rats, and rhesus
monkeys have suggested that cocaine is capable
of altering D2-like receptor levels within a few
hours of the last cocaine administration (Bailey
et al. 2008; Le Foil et al. 2005; Maggos et al.
1998; Nader et al. 2002; Nader et al. 2006;
Tsukada et al. 1996), it is important to note
that differences in the dosing regimen, route of
administration, and length of time since cocaine
treatment are all known to affect changes in
D2-like receptor density (e.g., Anderson and
Pierce 2005). Consistent with the
cocaine-induced changes in pramipexole-induced
yawning that developed over the course of
the study, significant increases in D3 receptor
binding, as well as significant decreases in
D2-like receptor density and increases in
D2-like receptor affinity were observed in the
ventral striatum of cocaine-treated rats 42 days
after the cessation of cocaine. Not only are
these cocaine-induced changes in D3 receptor
binding similar to the time-dependent increases
in D3 receptor binding and cell surface
expression that have been reported following
periods of cocaine self-administration in rats
(Conrad et al. 2010; Neisewander et al. 2004),
but the observed decreases in D2-like receptor
density are also similar to recent reports of
decreases in D2 receptor expression in rats with
a history of cocaine self-administration (Conrad
et al. 2010). It should be noted, however, that
similar decreases in D2 and increases in D3
receptor densities have also been reported
following the repeated administration of high
doses of D2-like agonists (Stanwood et al.
2000). Although an interaction between cocaine
pretreatment and pramipexole cannot be ruled
out, the fact that repeated pramipexole
administration failed to alter the binding
properties of either D2-like or D3 receptors in
the saline-treated group suggests that the
observed changes in D2-like and D3 receptor
binding resulted from the repeated
administration of cocaine, and not
pramipexole.

Curiously, unlike the rapid sensitization of
rats to the D2mediated effects of pramipexole
that has been observed with food restriction
(Collins et al. 2008), cocaine treatment
appeared to have a delayed effect on the
D2-mediated effects of pramipexole as no changes
in the sensitivity of rats to the inhibition of
yawning, induction of hypothermia, and
locomotor stimulation were observed throughout
the first 21 days after the cessation of cocaine
administration. Importantly, however, increases
in the sensitivity of rats to the inhibitory
effects of pramipexole on yawning
corresponded to a significant decrease in Kd for
[3H]spiperone at D2-like receptors at 42
days, suggesting that the observed behavioral
changes were mediated by an increased affinity
of pramipexole at the D2 receptor. Although the
current studies were not designed to
differentiate between the highand low-affinity
states of D2 receptors, it is possible that the
increased sensitivity of the cocaine-treated
rats to the D2mediated inhibition of
pramipexole-induced yawning was also
influenced by an increased proportion of D2
receptors in the high-affinity state as has been
reported in rats following cocaine
self-administration (Bnand et al. 2008) or
sensitization to caffeine (Simola et al. 2008)
or amphetamine (Seeman 2009). In addition to
providing convergent evidence to support a
cocaine-induced sensitization of the D2
receptor, the current studies also suggest that
increases in D3 receptor binding may underlie
the progressive increase in the sensitivity of
cocaine-treated rats to the D3-mediated
induction of yawning by low doses of
pramipexole. However, the limitations of
single-point assays make it impossible to
determine whether these behavioral changes
resulted from an increase in D3 receptor
density, D3 receptor affinity, or some
combination of the two. Interestingly, Hamilton
et al. (2010) have recently reported a similar
enhancement of D2-like agonist-induced
yawning in male monkeys that were exposed
to cocaine in utero. Not only was this effect
positively correlated with the maximal daily
dose of cocaine received during gestation, but
this enhanced yawning response was
observed 13 years later, suggesting that cocaine
exposure may result in long lasting, if not
permanent enhancements of the function and/or
sensitivity of D3 receptors.

In addition to altering the potency and/or
effectiveness of drugs (e.g., psycho stimulants
or D2-like agonists) to stimulate locomotor
activity or elicit other behavioral effects, the
neuroadaptations that underlie behavioral
sensitization are also thought to play a crucial
role in the development of addiction-like
behaviors in animals (e.g., Kalivas and Stewart
1991; Robinson and Berridge 1993; Vezina 2004),
such as the increased capacity of cocaine and
direct D2-like agonists to increase responding
that was previously reinforced by cocaine (De
Vries et al. 1998; De Vries et al. 2002; Dias et
al. 2004; Edwards et al. 2007). Although these
studies suggest that increases in responding are
mediated by a sensitization of D2-like receptors
in general, there is growing evidence to support
a role for the D3 receptor, specifically in the
capacity of cocaine-paired cues to reinstate or
maintain responding following periods of cocaine
self-administration (Cervo et al. 2007; Collins
and Woods 2009; Gal and Gyertyan 2006; Gilbert
et al. 2005). Given this putative role for the
D3 receptor, it is interesting to note that the
capacity of these cues to reinstate responding
appears to be positively correlated with the
duration of abstinence insofar as progressive
and persistent increases in responding have been
observed over the first 3 months of abstinence
(e.g., Grimm et al. 2001; Lu et al. 2004). This
effect appears to be similar in magnitude and
time course to the increases in D3-mediated
yawning observed in the current
studies.

In summary, these studies provide convergent
behavioral and molecular evidence to support a
cocaine-induced enhancement of both D3- and
D2-mediated elicited behavioral effects in rats.
The temporal differences in the development of
these effects suggest that increases in the
sensitivity of rats to the D3-mediated effects
of pramipexole emerge as early as 72 h after the
cessation of cocaine administration with
progressive and persistent enhancements observed
for at least 42 days, whereas increases in the
sensitivity of rats to the D2-mediated
behavioral effects of pramipexole do not become
apparent until after longer periods of time have
passed. In addition to suggesting that D2-like
agonist-induced yawning may provide a
valuable tool for the evaluation of the
functional changes in D3 and D2 receptors that
occur following cocaine administration, when
taken together with previous reports (Conrad et
al. 2010; Hamilton et al. 2010; Neisewander et
al. 2004), these studies provide further support
for the notion that the progressive and
persistent increases in the density, function,
and/or sensitivity of the D3 receptor that occur
during periods of abstinence or withdrawal may
contribute to the enduring vulnerability to
relapse commonly observed in human drug
abusers.